Adenocarcinoma +++ ++ + Papillary Adenocarcinoma +++ ++ +
Total Page:16
File Type:pdf, Size:1020Kb
A Adenocarcinoma Total case no. +++ ++ + ─ Positive (%) HPC1 0 6 13 8 70.37 HPC2 1 2 9 15 44.44 27 HPC4 5 18 2 2 92.59 Con phage 0 0 0 27 0.00 Papillary adenocarcinoma Total case no. +++ ++ + ─ Positive (%) HPC1 2 1 3 2 75.00 HPC2 0 1 3 4 50.00 8 HPC4 1 5 1 1 87.50 Con phage 0 0 0 8 0.00 Bronchioloalveolar carcinoma Total case no. +++ ++ + ─ Positive (%) HPC1 2 4 1 1 87.50 HPC2 0 0 1 7 12.50 8 HPC4 1 5 1 1 87.50 Con phage 0 0 0 8 0.00 Squamous cell carcinoma Total case no. +++ ++ + ─ Positive (%) HPC1 5 7 12 3 88.89 HPC2 0 2 7 18 33.33 27 HPC4 9 16 1 1 96.30 Con phage 0 0 0 27 0.00 Large cell carcinoma Total case no. +++ ++ + ─ Positive (%) HPC1 0 6 3 1 90.00 HPC2 0 0 4 6 40.00 10 HPC4 5 4 0 1 90.00 Con phage 0 0 0 10 0.00 Small cell carcinoma Total case no. +++ ++ + ─ Positive (%) HPC1 2 5 1 0 100.00 HPC2 0 0 2 6 25.00 8 HPC4 5 3 0 0 100.00 Con phage 0 0 0 8 0.00 1 B Metastatic adenocarcinoma from lung Total case no. +++ ++ + ─ Positive (%) HPC1 0 1 6 1 87.50 HPC2 0 0 3 5 37.50 8 HPC4 2 4 2 0 100.00 Con phage 0 0 0 8 0.00 Metastatic squamous cell carcinoma from lung Total case no. +++ ++ + ─ Positive (%) HPC1 0 2 0 2 50.00 HPC2 0 0 1 3 25.00 4 HPC4 2 2 0 0 100.00 Con phage 0 0 0 4 0.00 C Normal pneumonic tissue Total case no. +++ ++ + ─ Positive (%) HPC1 0 0 0 6 0.00 HPC2 0 0 0 6 0.00 6 HPC4 0 0 0 6 0.00 Con phage 0 0 0 6 0.00 Cancer adjacent normal pneumonic tissue Total case no. +++ ++ + ─ Positive (%) HPC1 0 0 0 6 0.00 HPC2 0 0 0 6 0.00 6 HPC4 0 0 0 6 0.00 Con phage 0 0 0 6 0.00 2 Table S1. Clinical response rates in human lung cancer surgical specimens, as evidenced by immunohistochemistry (IHC) using HPC1, HPC2, and HPC4. (A) Several histopathological subtypes of clinical human lung cancer biopsies were immunostained by HPC1, HPC2, or HPC4, and compared to control phage. The positive response percentages were calculated and compiled. (B) IHC data for metastatic adenocarcinoma and SCC from lung. (C) Tumor specific binding of HPC1, HPC2, and HPC4 were examined using normal pneumonic tissue and cancer adjacent normal pneumonic tissue. Reaction area: +++, >50%; ++, 50~20%; +, <20%; -, 0%. 3 A Adenocarcinoma Grade Stage Type Con phageHPC1 HPC2 HPC4 AD1 1 T2N1M0 Malignant െ ++ െ +++ AD2 1 T2N1M0 Malignant െ + െ ++ AD3 1 T4NxM0 Malignant െ െ + + AD4 2 T2N1M0 Malignant െ ++ + +++ AD5 2 T1N0M0 Malignant െ ++ + ++ AD6 - T1N0M0 Malignant െ + െ +++ AD7 1 T3N1M0 Malignant െ + + ++ AD8 2 T1NxM0 Malignant െ + െ ++ AD9 1 TxN0M0 Malignant െ + െ ++ AD10 1 T4N1M0 Malignant െ + + ++ AD11 1 T1NxM0 Malignant െ െ െ ++ AD12 2 T4N2M0 Malignant െ െ + ++ AD13 2 T3N1M0 Malignant െ െ െ െ AD14 2 T3N1M0 Malignant െ െ െ െ AD15 2 T3N1M1 Malignant െ െ െ ++ AD16 3 T2N0M0 Malignant െ + ++ ++ AD17 2 T2N0M1 Malignant െ + +++ ++ AD18 2 T2N0M0 Malignant െ ++ െ ++ AD19 2 T2N0M0 Malignant െ + ++ +++ AD20 2 T2N1M0 Malignant െ െ + ++ AD21 3 T4NxM0 Malignant െ + െ ++ AD22 2 T3N0M0 Malignant െ + + ++ AD23 3 T4N0M0 Malignant െ ++ + +++ AD24 3 T3N0M0 Malignant െ െ െ ++ AD25 3 T1N0M0 Malignant െ ++ െ ++ AD26 3 T2N0M0 Malignant െ + െ ++ AD27 3 T2N0M0 Malignant െ + െ + 4 B Papillary adenocarcinoma Grade Stage Type Con phageHPC1 HPC2 HPC4 PAD1 1 T3N0M0 Malignant െ + + ++ PAD2 1 T2NxM0 Malignant െ + ++ +++ PAD3 2 T1N0M0 Malignant െ െ + ++ PAD4 2 T2N0M0 Malignant െ + + ++ PAD5 1 T1N0M0 Malignant െ െ െ െ PAD6 2 T1N0M0 Malignant െ +++ െ ++ PAD7 2 - 3 TxNxMx Malignant െ ++ െ + PAD8 2 T2N0M0 Malignant െ +++ െ ++ C Bronchioloalveolar carcinoma Grade Stage Type Con phageHPC1 HPC2 HPC4 BAC1 - T2N0M0 Malignant െ ++ െ ++ BAC2 - T3N0M0 Malignant െ ++ െ ++ BAC3 - T2N0M0 Malignant െ + െ ++ BAC4 - T2N0M0 Malignant െ +++ + ++ BAC5 - T2N0M0 Malignant െ ++ െ + BAC6 - T1N0M0 Malignant െ ++ െ +++ BAC7 - T2N0M0 Malignant െ െ െ െ BAC8 - TxNxMx Malignant െ +++ െ ++ 5 D Squamous cell carcinoma Grade Stage Type Con phageHPC1 HPC2 HPC4 SCC1 1 T2N0M0 Malignant െ ++ + +++ SCC2 1 T1NxM0 Malignant െ + െ ++ SCC3 1 T2N1M0 Malignant െ + ++ ++ SCC4 1 T2N3M0 Malignant െ + + ++ SCC5 1 T3N1M0 Malignant െ ++ + +++ SCC6 1 T2N1M0 Malignant െ + െ ++ SCC7 1 T2N1M0 Malignant െ ++ + +++ SCC8 1 T2NxM0 Malignant െ ++ െ +++ SCC9 1 T2N0M0 Malignant െ + െ +++ SCC10 1 T4NxM1 Malignant െ െ െ െ SCC11 1 T3N1M0 Malignant െ ++ െ ++ SCC12 2 T3N0M0 Malignant െ +++ + ++ SCC13 1 T3N1M0 Malignant െ + ++ +++ SCC14 2 T2N0M0 Malignant െ + െ ++ SCC15 2 T2N0M0 Malignant െ + െ ++ SCC16 2 T2N1M0 Malignant െ െ െ + SCC17 2 T3N3M0 Malignant െ + + ++ SCC18 3 T2N0M0 Malignant െ + െ ++ SCC19 2 T3N1M0 Malignant െ + െ ++ SCC20 2 T1N0M0 Malignant െ +++ െ ++ SCC21 3 T3NxM0 Malignant െ +++ െ ++ SCC22 3 T1N0M0 Malignant െ ++ െ +++ SCC23 3 T2N0M0 Malignant െ ++ െ +++ SCC24 3 T2N0M0 Malignant െ +++ െ +++ SCC25 3 T3N3M0 Malignant െ + െ ++ SCC26 3 T3N1M0 Malignant െ െ + ++ SCC27 3 T2N0M0 Malignant െ +++ െ ++ 6 E Large cell carcinoma Grade Stage Type Con phageHPC1 HPC2 HPC4 GCC1 - T2N0M0 Malignant െ ++ + +++ GCC2 - T3N0M0 Malignant െ ++ + +++ LCC1 - T3N0M0 Malignant െ ++ െ ++ LCC2 - T2NxM0 Malignant െ ++ + +++ LCC3 - T3N1M0 Malignant െ + െ ++ LCC4 - T2NxM0 Malignant െ + െ ++ LCC5 - T3NxM0 Malignant െ ++ + +++ LCC6 - T3N1M0 Malignant െ + െ ++ LCC7 - T2N0M0 Malignant െ ++ െ +++ LCC8 - T3NxM0 Malignant െ െ െ െ F Small cell carcinoma Grade Stage Type Con phageHPC1 HPC2 HPC4 SmCC1 - T3N1M0 Malignant െ +++ െ +++ SmCC2 - T3N2M0 Malignant െ + െ ++ SmCC3 - T2N1M0 Malignant െ ++ െ +++ SmCC4 - T3NxM0 Malignant െ ++ െ +++ SmCC5 - T2N1M0 Malignant െ ++ െ +++ SmCC6 - T3N0M0 Malignant െ ++ + +++ SmCC7 - T2N0M0 Malignant െ ++ െ ++ SmCC8 - T2N0M0 Malignant െ +++ + ++ 7 Table S2. Pathological diagnostic information of lung cancer stage and grade and their correlation to HPC1, HPC2, and HPC4 response rates. Reaction area: +++, >50%; ++, 50~20%; +, <20%; -, 0%. 8 A B Figure S1. In vitro phage display biopanning and selection of phage clones that bind to NSCLC cell lines. (A) A phage displayed random peptide library was used to select phages that bind to the lung large cell carcinoma cell line H460. (B) Forty-seven H460 bound phage clones with higher binding ability to NCI-H1993, CL1-5, A549, and 3LL cell lines were selected from the fifth round of biopanning for ELISA screening. Helper phage was used as a negative control. NNM was used as the normal cell control. 9 A B Figure S2. Predicted motifs and lung cancer cell line binding patterns of two major phage clone groups. (A) Binding intensity of HPC2, HPC3, HPC4, and HPC6 to H460, H1993, CL1-5, A549, and 3LL, as measured by ELISA of paraformaldehyde-fixed cells. The binding patterns of HPC3, HPC4, and HPC6 to these cell lines are similar, and distinct from those of HPC2. These data, combined with the observation that HPC4 exhibits better response rates in clinical biopsy and other cell-level experiments, suggest that the W-EMM mimetic motif plays a more important role than the NPW motif in human lung cancer binding. (B) HPC1, HPC12, HPC10, and HPC9 show similar binding patterns, suggesting that the MHL-W consensus sequence contributes to such binding. 10 A H460 H1993 FITC DAPI Merge FITC DAPI Merge HSP1-FITC HSP2-FITC HSP4-FITC ConP-FITC B Figure S3. Determination of binding specificity of FITC-labeled HSP1, HSP2, and HSP4 peptides to H460 and H1993 cells by immunofluorescent staining. (A) Immunofluorescent staining of FITC-labeled HSP1, HSP2, and HSP4 peptides to H460 large cell carcinoma and H1993 adenocarcinoma cell lines. Nuclei were stained with DAPI. Scale bar, 50 μm. (B) Table listing the percentage of IFA-positive cells for HSP1, HSP2, and HSP4-FITC in H460 and H1993 cell lines. 11 FITC DAPI Merge HSP1-FITC ScP1-FITC HSP2-FITC ScP2-FITC HSP4-FITC ScP4-FITC Figure S4. Scrambled peptides show no binding ability to H460 cells. Immunofluorescent staining of FITC-labeled scrambled peptides ScP1 (LGHPMATMWLGH), ScP2 (MYQEPSRWGENY), and ScP4 (IMEWNEYIMRPN) corresponding to HSP1, HSP2, and HSP4 for H460 binding. The nuclei were stained with DAPI. Scale bar, 20 μm. 12 Figure S5. A Large Cell Squamous Cell Small Cell Adenocarcinoma Carcinoma Carcinoma Carcinoma HPC2 HPC4 Con phage HPC1 B Pseudostratified Normal Normal Alveoli Columnar Bronchiole Epithelium HPC2 HPC4 Con phage HPC1 13 Figure S5. Immunohistochemical staining of human NSCLC, SCLC, and normal pneumonic tissues with HPC1, HPC2, and HPC4. Lower magnification (larger field of view) images, comparable to those shown in Figure 1B. (A) Serial sections of four subtypes of NSCLC and SCLC revealed prominent staining in tumor regions by HPC1 and HPC4, but weak or no signals in stroma regions. (B) Three histological types of tissue in normal lung did not cross-react with HPC1, HPC2, or HPC4. The same phage titer (2~5 × 108 pfu/μl) was used in each type of tissue. Helper phage was used as a negative control. Scale bar, 100 μm. 14 Figure S6. Phage distribution of the liver, kidney, and spleen in tumor-homing experiments. Although there was a noticeable uptake of phage by the organs of mononuclear phagocyte system such as the liver and spleen, no significant difference in accumulation between the targeting and control phages was observed in these organs. 15 Figure S7. In vitro cell binding of targeting SPIONs. Prussian blue staining of fixed H460 cells treated with HSP1-, HSP2-, HSP4-, Ctrl P-Dex- Fe3O4,orDex-Fe3O4 nanoparticles (10 μg/ml) for 1 hr. 16 Figure S8. HSP1, HSP2, and HSP4 peptides enhance LSRB in a human lung cancer cell line, H460. Kinetics of HSP1-LSRB, HSP2-LSRB, HSP4-LSRB, and LSRB uptake by H460 cells at 37°C. After washing with acid glycine buffer to remove surface-bound liposomal dye, internalized SRB was quantified (n=4). 17 A B Figure S9. HSP1, HSP2, and HSP4 peptides enhance LSRB internalization and LD uptake in a human lung adenocarcinoma cell line, H1993. (A) Uptake kinetics of non-targeting LSRB and peptide (HSP1, HSP2, and HSP4)- conjugated LSRB by H1993 cells at 37°C. After washing with acid glycine buffer to remove surface-bound liposomal dye, internalized SRB was quantified (n=4). These results indicate that HSP4 is most effective at triggering endocytosis in the H1993 adenocarcinoma cell line from low to high concentrations; notably, this kinetic pattern is distinct from that observed using H460 large cell carcinoma cell lines (Figure S8).